Types of Cancer	Expressed Antigens

Breast	Her-2 (human epithelial growth factor
	receptor type 2)
	Ep-CAM (epithelial cell adhesion molecule)
Prostate	PSCA, Sigma Receptor, CD-44, Transferrin
	Receptor
Liver
Colon and Rectum	Ep-CAM (epithelial cell adhesion molecule)
	Transferrin Receptor
	Carcinoembryonic antigen (CEA)
Urinary Bladder
Non-Hodgkin Lymphoma
Melanomas of the skin	Mart-1, GM-2
Kidney and Renal Pelvis
Pancreas	Mesothelin
Oral Cavity and Pharynx
Ovary	Folate Receptor, CA-125, Mesothelin

EXAMPLE 1Effects of Zinc on PC12 Cells

The addition of zinc to PC12 cells in vitro leads to time and dose-dependent cell death. This occurs with either undifferentiated or differentiated PC12 cells. In both cases, within 5 hours, cell death was accompanied by the progression to a more rounded morphology, shrinkage in overall cell size, and membrane blebbing, which is indicative of apoptosis. With the higher zinc concentrations (<100 μM), or longer time periods, significant cell lysis is also observed, which is indicative of necrosis, a novel observation for zinc .

When the PC12 cells were preincubated with the MEK inhibitor, U1026, zinc-induced cell death was greatly attenuated. Under those conditions, zinc cytotoxicity occurred with a delayed time-course, requiring overnight incubations to observe significant cell death. Under such circumstances, a low percentage of the cell populations underwent morphological changes associated with cell death, with cell lysis (an indicator of necrosis) predominating. Preincubation of these cells with inhibitors of the other members of the MAP kinase signaling cascades had no effect on zinc-mediated cell death. The data suggest that ERK mediates the cytotoxic effect of zinc, while JNK and p38 are not involved. Likewise, zinc treatment of PC12 cells induced ERK activation, while preincubation of cells with U1026 attenuated that activation. pERK could be detected within five minutes of treatment and remained at that level shown for at least five hours, which was the longest time period examined in this example.

These cytotoxic effects observed in PC12 cells (supra) are attributed to zinc. While ZnCl₂was used in initial experiments, ZnSO4 was equally effective as ZnCl₂. No cell death, neither apoptosis nor necrosis, was observed when incubations include NiCl₂, CoCl₂or CuSO₄at the same range of the concentrations as zinc. Interestingly, Cd treatment did result in cell death, but through an ERK-independent mechanism. High (mM) concentrations of Mg, Ca, Mn or Li had no effect on cell viability. Similar experiments with IIC9 cells produced the same results.

EXAMPLE 2Increased Intracellular Zinc Induces Cell Death In Multiple Cell Types

Exemplary cell types were treated in vitro with varying amounts of zinc (zinc chloride or zinc sulfate), in the presence or absence of the zinc ionophore pyrithione. As is depicted in tables 2 and 3, lower concentrations of zinc are effective in killing cells when the zinc ionophore is present, indicating that zinc function intracellularly. Cell death involves both apoptosis and necrosis as determined by morphological observation.

TABLE 2


Zinc-induced death of RAW (macrophage) cells

Treatment

Hours of	25μM	25 μM +	50μM	50 μM +	100 μM
Incubation	zinc	pyr	zinc	pyr	zinc

0.0	0.0	0.0
1.0	0.0	0.0
3.0	0.0	15.0
5.0	0.0	35.0	0.0	50.0
18.0			20.0		100.0

pyr = pyrithione (zinc ionophore); numbers = % cell death

TABLE 3


Zinc-induced death of N18TG2 (neuroblastoma) cells
24 h incubation

	Treatment	% death

10	μM zinc + pyr	86
25	μM zinc	19
25	μM zinc + pyr	87
50	μM zinc	75
75	μM zinc	84
150	μM zinc	90

EXAMPLE 3Zinc Kills IIC9 Cells in an ERK-Dependent Manner

In this example and the following examples, we show that zinc, (as in other cell lines such as PC12, RAW macrophages, and N18TG2 neuroblastoma cells) induces the apoptotic death of IIC9 embryonic fibroblasts. As with PC12 cells, death is ERK-dependent. Unlike previous examples, however, IIC9 cells are p53 minus. Thus zinc-mediated apoptosis occurs via a p53-independent mechanism. Death is also facilitated by the presence of the zinc ionophore, pyrithione, indicating that intracellular zinc initiates the death response. To understand the upstream activator of zinc-induced ERK1/2 activation, we examined the role of Ras in this process. Expression of dn-(dominant negative) Ras attenuates ERK1/2 activation by zinc and correspondingly reduces its cytotoxic effects. Raf-RBD pull-down experiments were used to confirm that zinc treatment activates Ras and to identify the specific Ras isoform that is activated. Importantly, zinc uniquely activates H-Ras. This contrasts the activation of K- and N-Ras that occurs when IIC9 cells are stimulated by mitogen. Thus, although the kinetics of activation of the Ras/ERK pathway are similar to those seen when induced by mitogen, the distinguishing feature appears to be the isoform specificity of Ras activation.

Cells and Incubation Conditions: IIC9 cells were grown in DMEM containing 10% FCS, P/S. Cells were plated in 12-well plates and grown to approximately 70% confluency. At that time, cells were serum arrested by changing the medium to DMEM (low glucose) without FCS. After 48 hours, cells were treated with zinc as described in the text. Cell death was quantified by trypan blue uptake. Cells were incubated with trypan blue (0.04%) for 5 min. Cells were examined microscopically and the number of blue cells and total cells were counted. Four fields were examined for each condition, with a minimum of 100 cells/field. To examine the effects of selective inhibitors, cells were preincubated with the indicated compound for 30 min. prior to zinc treatment.

Assays: ERK1/2 activation was determined by harvesting cells directly in boiling SDS-PAGE sample buffer and probing corresponding Western Blots with anti-pERK antibody (Cell Signaling). Blots were stripped and reprobed for total ERK (Cell Signaling). Ras activation was determined using the Raf-RBD assay (21,24). Identification of the specific Ras isoform activated was determined by probing the western blot with isoform-specific antibody (Upstate Biotechnology). Transfection of IIC9 cells was carried out using Novafector. Transfection efficiency of these cells is approximately 90%. The constructs for expression of dn-Ras and Bcl-Xl were kindly provided by Dr. J. Corbett and Dr. Chinnaduri (SLU).

The addition of zinc to IIC9 cells led to cell death in a time- and concentration-dependent manner. Little or no death was observed during the first few hours of cell incubation with zinc concentrations as high as 200 μM (FIG. 1A). However, during this time frame, zinc does induce dramatic changes in cell morphology. In response to either 100 or 200 uM zinc, all cells were rounded and underwent differing degrees of cell shrinkage (FIG. 1C). This contrasts their normal, irregular, and somewhat elongated morphology (FIG. 1B) In response to 50 μM zinc , the lowest concentration used in this particular experiment, approximately 50% of the cell population display a rounded morphology at this time. After a 6 h period, cell death is evident, with the percentage of non-viable cells being relative to the concentration of zinc present (FIG. 1A). In this experiment, cell death was approximately 45%, 25%, and 15% when cells were treated with 200 μM, 100 μM and 50 μM zinc for 6 h respectively. Lysed cells could also be seen as a result of treatment with the higher zinc concentrations (FIG. 2D). With longer incubation times, the percentage of non-viable, cells continued to increase (FIG. 1B) and cell lysis became more predominant even at the lower zinc concentrations.

The effects observed are specific for zinc. ZnSO₄is equally effective as ZnCl₂, indicating that zinc is the effective component. Treatment of cells with NiCl₂, CoCl₂or CuSO₄at the same range of concentrations as zinc does not result in the cell death of IIC9 cells. High (mM) concentrations of Mg, Ca, Mn or Li have no effect on cell viability.

JNK and p38 are well known effectors of stress signals, with established roles in apoptosis. These two members of the MAPK family, however, are not mediators of zinc-induced cytotoxicity since preincubation of cells with SP600125 or SB203580, well characterized selective inhibitors of JNK and p38 respectively, does not affect zinc-mediated cell death (FIG. 2A). In contrast, preincubation of IIC9 cells with U0126, an inhibitor of ERK activation, inhibits zinc-induced cell death. In the experiment shown, a 6-h incubation with 50 μM zinc leads to the death of approximately 25% of the population. Little or no death results when this concentration of zinc is added to cells pretreated with U0126.FIG. 2B-D shows that the morphological changes induced by zinc are also limited in cells preincubated with U0126.

Because blocking ERK activation attenuates zinc-mediated cell death, we reasoned that zinc treatment results in ERK activation. This was demonstrated by incubating IIC9 cells with 50M zinc and determining the presence of pERK by Western blotting with anti-pERK antiserum (FIG. 2E). ERK activation is rapid, detected within 5 min of zinc addition and remains elevated for at least 6 h, the longest time period examined (data not shown). Consistent with its effect on zinc cytoxicity, U0126 significantly reduces ERK activation by zinc. In the experiment shown, some pERK could be detected with longer exposure of the blot. Thus, it is possible that the residual ERK activity accounts for the ability of zinc to kill cells when it is present for the more extensive time periods. Alternatively, zinc may induce an additional death-pathway that is not ERK dependent.

EXAMPLE 4Zinc-Mediated Cell Death Occurs via Regulated Apoptosis and Necrosis

The initial morphological changes seen upon zinc addition are suggestive of apoptotic cell death (FIG. 1). To confirm the importance of the apoptotic pathway, we preincubated cells with Z-VAD, a general caspase inhibitor. As shown inFIG. 3A, addition of Z-VAD attenuated cell death. Cells treated with 50 μM zinc showed little cell death when pretreated with Z-VAD during the first 6 hours of zinc treatment. When cells are incubated with 100μM zinc, Z-VAD attenuates cell death by approximately 50 %. With longer time periods (21 h), the effect of Z-VAD was less dramatic. Cell death as a result of 50 μM zinc treatment was attenuated by 50% but limited attenuation was observed with 100 μM zinc. While not wishing to be bound by theory, those data can be explained by the possibility that Z-VAD does not completely inhibit caspases in IIC9 cells over this time period. Alternatively, zinc may induce both caspase-dependent (apoptotic) and caspase-independent (necrotic) cell death pathways, as suggested by the morphological changes that occur with the longer incubation times.

The processes involved in zinc-mediated cell death were further investigated by over-expressing Bcl-XL in IIC9 cells. Expression of Bcl-XL prevents zinc-mediated cell death even when IIC9 cells are incubated with concentrations of zinc as high as 200 μM (FIG. 3B-D). As shown, Bcl-XL expressing cells retain their normal morphology and do not undergo rounding, skrinkage, or other signs characteristic of apoptosis (FIG. 3D). This was true even after 24 h of treatment of IIC9 cells with 200μM zinc. Interestingly, over-expression of Bcl-Xl also eliminates any signs of cell lysis. This is a completely novel observation. The data indicate that over-expression of Bcl-XL prevents both the zinc-mediated apoptotic and necrotic pathways.

EXAMPLE 5Mechanism of Erk Activation

Previous studies have shown that zinc activates ERK1/2 in human epidermoid A431 cells and B82L parental fibroblasts in a manner that is blocked by inhibition of the EGF R and/or c-Src(45). In those cases, it appears that zinc stimulates a c-Src dependent phosphorylation of the EGF R. This trans-activation leads to enhanced ERK activity, and in the case of B82L cells, enhanced production of inflammatory cytokines. The effects of zinc on cell viability were undetermined. In contrast to those observations, treatment of IIC9 cells with PD153035 (EFG R inhibitor) does not alter ERK activation by zinc (FIG. 4A). Inhibition of the PDGF R or treatment of IIC9 cells with PP2 to inhibit Src does not inhibit zinc activation of ERK1/2 (data not shown). Extracellular zinc has been shown to activate ERK via a cell surface “zinc-sensing” receptor(3) To gain insight into the likelihood that zinc functions at the level of a cell surface receptor, we compared the effects of zinc treatment in the absence or presence of the heavy metal ionophore, pyrithione. In the presence of pyrithione, ERK activation is observed at markedly lower zinc concentrations than in its absence (FIG. 4B). Consistent with the enhanced ability of zinc to activate ERK when pyrithione is present, cell death also occurs at lower zinc concentrations (FIG. 4C). These data suggest that the immediate target for zinc-activation of the ERK1/2 signaling cascade that initiates a cell death response is intracellular.

Since zinc functions intracellularly, the time and concentration dependence of cell death seen in our experiments likely reflect the conditions necessary for cells to accumulate sufficient levels of zinc to induce the death pathway. For IIC9 cells, extracellular zinc concentrations of 50 μM or higher are necessary to do so, in the absence of pyrithione. Such levels are found in a number of tissues e.g. prostate, brain, pancreas (2,9,12,19,30) In brain, when conditions of excitotoxicity lead to sustained release of zinc from a subset of glutamenergic neurons, extracellular concentrations higher than 300 μM have been reported. Under such circumstances, transport of zinc into the post-synaptic cell is associated with its death (2,9).

EXAMPLE 6Zinc Activate ERK via a Specific Ras

Because Ras is a common upstream activator of ERK1/2 (36), we reasoned that zinc may induce the phosphorylation of ERK1/2 via Ras. This was examined by incubating IIC9 cells with 100 uM zinc and assessing Ras activation by RBD pull-down of GTP-Ras followed by Western blot analysis with a pan-Ras antibody. The results show that zinc induces approximately a 5-fold activation of Ras (FIG. 5A). The critical role of Ras in mediating the effects of zinc was confirmed by examining the effect of over-expression of dnRas. Expression of dn-Ras inhibits ERK activation, indicating that the Ras-Mek pathway is the predominant mechanism used by zinc to activate ERK. Consistent with Ras as the upstream activator of the cytotoxic effect of zinc on IIC9, expression of dn H-Ras also attenuates zinc-mediated cell death (FIG. 5B).

IIC9 cells express H-, N- and K-Ras. Previously we found that stimulation of IIC9 cells with thrombin results in an approximate 3- to 5-fold activation of Ras. Our more recent studies show that thrombin induces a rapid activation of N-Ras, followed by a sustained activation of K-Ras. Because thrombin and zinc activation of Ras in IIC9 cells result in different biological outcomes, we asked whether they activate distinct Ras isoforms. In contrast to the results obtained when cells are stimulated with thrombin, zinc stimulation results in the selective activation of H-Ras.

EXAMPLE 7Production of Liposomes Containing Zinc

To ensure delivery of relatively high concentrations of zinc to tumor cells, we developed a targeted encapsulated liposome delivery method. Liposomes, spherical, self-enclosed vesicles composed of amphipathic lipids, have been widely studied and are employed as vehicles for in vivo administration of therapeutic agents. In particular, the so-called long circulating liposomes formulations which avoid uptake by the organs of the mononuclear phagocyte system, primarily the liver and spleen, have found commercial applicability. This method has several advantages: 1) it allows for the delivery of high therapeutic concentrations of zinc, 2) only the targeted cells are exposed to high zinc concentrations. This effectively should increase the therapeutic range of zinc and finally 3) by encapsulating zinc within long circulating liposomes formulations containing polyethyleneglycol (PEG-liposome) the effective half life of the zinc can be effectively increased.

The first issue concerning the encapsulation of high concentrations of zinc was addressed by employing an ethanol injection method employing a complex mixture of neutral and positively charged phospholipids plus cholesterol (infra). The inventor envisions that liposomes may contain polyethylene glycol (PEG).

The second issue is to devise a method to achieve site specific delivery of the zinc containing liposomes. We choose to employ an approach that utilizes targeting ligands. The advantage of this method is that the method can be easily adapted to target different types of cells by altering the targeting ligand. For example, an antibody can be attached to the liposomes' surfaces. We choose an approach in which the targeting ligand is attached to the free ends of the polymer chains forming the surface coat on the liposomes (Allen. T. M., et al., Biochim. Biophys. Acta, 1237:99-108 (1995); Blume, G., et al., Biochim. Biophys. Acta, 1149:180-184 (1993)).

We choose the prostate cancer cell line PC3 cells to demonstrate that therapeutic concentrations of zinc (“killing”) can be delivered to cancer cells. These cells express high levels of the transferrin receptor and can be targeted by covalently attaching transferrin to the formed liposomes.

We next tested the ability of these liposomes to kill PC3 cells in culture (FIG. 6). Treatment of PC3 cells with liposomes that contained high concentrations of zinc and were covalently linked to transferrin resulted in effective cell death. In contrast, liposomes that either did not encapsulate zinc or did not contain transferrin were ineffective (FIG. 6).

In a prophetic liposome, PEG is incorporated into our liposomes to ensure that our liposome preparations have reduced opsinisation in the plasma. Thus, the liposomes exhibit reduced uptake by the reticuloendothelial system (RES), which primarily consists of macrophages in the liver and the spleen. Furthermore, these liposomes can preferentially accumulate in solid tumors via the increased permeability of the tumor endothelium and reduced lymphatic drainage, or enhanced permeability and retention (EPR) effect.

Non-prophetically, zinc containing liposomes were prepared by injecting ethanolic solutions of lipids into 10 mM Hepes buffer pH 6 containing 2.5 M ZnCl₂. To make the ethanolic lipid solution, lipids initially dissolved in chloroform were dried by passing gaseous nitrogen over the solution. The dried lipids were dissolved in ethanol at a concentration of 2 to 50 pmol of phospholipids per mL of alcohol. The alcoholic lipid solution containing dioleoyl phosphatidyly choline, 1,2 dioleoyl phosphatidyly ethanolamine, 1,2 dioleolyl phosphatidyletanolamine-N-4-(p-maleimido-phenyl)butyramide) and cholesterol in a ratio of 7:2:0.05:1 was slowly (0.5 ml/min) injected through a Hamilton syringe into 3 mL of a magnetically stirred Hepes buffer, containing 2.5 mM ZnCl₂The final concentration of ethanol was between 2.5-7.5%. During injection of the lipids, the solution was constantly stirred by a standard magnetic stirrer. To ensure that the temperature of the liposome solution was well above the phase transition of the lipids, the temperature was kept at 350 C. Because the transferrin coupling reaction is carried out in sodium borate buffer pH 8.5, the liposomes were dialyzed overnight against a 0.15 mM sodium borate, 0.1 mM EDTA buffer pH 8.5. Before coupling transferrin to the liposomes, 100 μg of transferrin was thiolated by treating transferrin with 400 nmoles of fresh Traut's reagent in 2 ml of the sodium borate buffer for 1 hour under dark conditions at room temp. To link the thiolated transferrin to the liposomes, thiolated transferrin was immediately added to the liposomes and left for 24 hrs at 37° C. After 24 hours the liposomes were dialyzed against 50 mM Tris buffer pH 7.2 and then briefly sonicated in a bath sonifier, for approximately 5 minutes.

Although several embodiments have been described in detail for purposes of illustration, various modifications may be made to each without departing from the scope and spirit of the invention. Accordingly, the invention is not to be limited, except as by the appended claims.